Spatial positioning of spectrally labeled beads

ABSTRACT

Devices, systems, kits, and methods for detecting and/or identifying a plurality of spectrally labeled bodies well-suited for performing multiplexed assays. By spectrally labeling the beads with materials which generate identifiable spectra, a plurality of beads may be identified within the fluid. Reading of the beads is facilitated by restraining the beads in arrays, and/or using a focused laser.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The application claims the benefit of priority from co-pendingU.S. Provisional Patent Application No. 60/195,520 entitled “Method forEncoding Materials with Semiconductor Nanocrystals, Compositions MadeThereby, and Devices for Detection and Decoding Thereof,” filed Apr. 6,2000, the full disclosure of which is incorporated herein by reference.

[0002] The subject matter of the present application is related to thefollowing co-pending patent applications, the disclosures of which arealso incorporated herein by reference: U.S. patent application Ser. No.09/160,458 filed Sep. 24, 1998 and entitled, “Inventory Control”; U.S.patent application Ser. No. 09/397,432 filed Sep. 17, 1999, and alsoentitled “Inventory Control”; PCT Patent Application No. WO 99/50916 aspublished on April 1, 1999, entitled “Quantum Dot White and ColoredLight Emitting Diodes”; and U.S. Patent Application No. 09/259,982 filedMar. 1, 1999, and entitled “Semiconductor Nanocrystal Probes forBiological Applications and Process for Making and Using Such Probes”.All other references cited herein are also incorporated by reference.

BACKGROUND OF THE INVENTION

[0003] The present invention generally provides devices, compositions ofmatter, kits, systems, and methods for detecting and identifying aplurality of spectrally labeled bodies. In a particular embodiment, theinvention provides systems and methods for detecting and identifying aplurality of spectral codes generated by such bodies, especially formeasuring the results of high-throughput bead-based assay systems, andthe like. The invention will often use labeled beads which generateidentifiable spectra that include a number of discreet signals havingmeasurable characteristics, such as wavelength and/or intensity.

[0004] Tracking the locations and/or identities of a large number ofitems can be challenging in many settings. Barcode technology ingeneral, and the Universal Product Code in particular, has provided hugebenefits for tracking a variety of objects. Barcode technologies oftenuse a linear array of elements printed either directly on an object oron labels which may be affixed to the object. These barcode elementsoften comprise bars and spaces, with the bars having varying widths torepresent strings of binary ones, and the spaces between the bars havingvarying widths to represent strings of binary zeros.

[0005] Barcodes can be detected optically using devices such as scanninglaser beams or handheld wands. Similar barcode schemes can beimplemented in magnetic media. The scanning systems oftenelectro-optically decode the label to determine multiple alphanumericalcharacters that are intended to be descriptive of (or otherwiseidentify) the article or its character. These barcodes are oftenpresented in digital form as an input to a data processing system, forexample, for use in point-of-sale processing, inventory control, and thelike.

[0006] Barcode techniques such as the Universal Product Code have gainedwide acceptance, and a variety of higher density alternatives also havebeen proposed. Unfortunately, these standard barcodes are oftenunsuitable for labeling many “libraries” or groupings of elements. Forexample, small items such as jewelry or minute electrical components maylack sufficient surface area for convenient attachment of the barcode.Similarly, emerging technologies such as combinatorial chemistry,genomics research, microfluidics, potential pharmaceutical screening,micromachines, and other nanoscale technologies do not appearwell-suited for supporting known, relatively large-scale barcode labels.In many of these developing fields, it is often desirable to make use oflarge numbers of compounds within a fluid, and identifying and trackingthe movements of such fluids using existing barcodes is particularlyproblematic. While a few chemical encoding systems for chemicals andfluids have been proposed, reliable and accurate labeling of largenumbers of small and/or fluid elements remains a challenge.

[0007] Small scale and fluid labeling capabilities have recentlyadvanced radically TM with the suggested application of semiconductornanocrystals (also known as Q-Dot™ particles), as detailed in U.S.patent application Ser. No. 09/397,432, the full disclosure of which isincorporated herein by reference. Semiconductor nanocrystals aremicroscopic particles having size-dependent optical and/orelectromagnetic emission properties. As the band gap energy of suchsemiconductor nanocrystals vary with a size, coating and/or material ofthe crystal, populations of these crystals can be produced having avariety of spectral emission characteristics. Furthermore, the intensityof the emission of a particular wavelength can be varied, therebyenabling the use of a variety of encoding schemes. A spectral labeldefined by a combination of semiconductor nanocrystals having differingemission signals can be identified from the characteristics of thespectrum emitted by the label when the semiconductor nanocrystals areenergized.

[0008] A particularly promising application for semiconductornanocrystals is in multiplexed and/or high-throughput assay systems.Multiplexed assay formats would be highly desirable for improvedthroughput capability, and to match the demands that combinatorialchemistry is putting on established discovery and validation systems forpharmaceuticals. For example, simultaneous elucidation of complexprotein patterns may allow detection of rare events or conditions, suchas cancer. In addition, the ever-expanding repertoire of genomicinformation would benefit from very efficient, parallel and inexpensiveassay formats. Desirable multiplexed assay characteristics may includeease of use, reliability of results, a high-throughput format, andextremely fast and inexpensive assay development and execution.

[0009] A number of known assay formats may currently be employed forhigh-throughput testing. Each of these formats has limitations, however.By far the most dominant high-throughput technique is based on theseparation of different assays into different regions of space. The96-well plate format is the workhorse in this arena.

[0010] In 96-well plate assays, the individual wells (which are isolatedfrom each other by walls) are often charged with different components,and the assay is performed and then the assay result in each wellmeasured. The information about which assay is being run is carried withthe well number or the position on the plate, and the result at thegiven position determines which assays are positive. These assays can bebased on chemiluminescence, scintillation , fluorescence, scattering, orabsorbance/colorimetric measurements, and the details of the detectionscheme depend on the reaction being assayed.

[0011] Multi-well assays have been reduced in size to enhancethroughput, for example, to accommodate 384 or 1536 wells per plate.Unfortunately, the fluid delivery and evaporation of the assay solutionat this scale are significantly more confounding to the assays.High-throughput formats based on multi-well arraying often rely oncomplex robotics and fluid dispensing systems to function optimally. Thedispensing of the appropriate solutions to the appropriate bins on theplate poses a challenge from both an efficiency and a contaminationstandpoint, and pains must be taken to optimize the fluidics for bothproperties. Furthermore, the throughput is ultimately limited by thenumber of wells that one can put adjacent on a plate, and the volume ofeach well. Arbitrarily small wells may have arbitrarily small volumes,resulting in a signal that scales with the volume, shrinkingproportionally with the cube of the radius. Nonetheless the spatialisolation of each well, and thereby each assay, has been much morecommon than running multiple assays in a single well. Such single-wellmultiplexing techniques are not widely used, due in large part to thedifficulty in “demultiplexing” or resolving the results of the differentassays in a single well.

[0012] For even higher throughput genomic and genetic analysistechniques, positional array technology has been shrunk to microscopicscales, often using high-density oligonucleotide arrays. Over a 1-cmsquare of glass, tens to hundreds of thousands of different nucleotidescan be written in, for example, 25-μm spots, which are well resolvedfrom each other. On this planar test structure or “chip,” which isemblazoned with an alignment grid, a particular spot's x,y positiondetermines which oligonucleotide is present at that spot. Typically 3′-or 5′-labeled amplified DNA is added to the array, hybridized and isthen detected using fluorescence-based techniques. Although this is avery powerful technique for assaying a large number of genetic markerssimultaneously, the cost is still very high, and the flexibility of thisassay is limited.

[0013] Once the masks have been written for the photolithographicprocess that builds the particular DNA sequences into a particularlocation on the chip, they are fixed and the addition thereto of newmarkers comes at a very high price. The extremely small feature size,and the highly parallel assay format, comes at the cost of theflexibility inherent in a common platform system such as the 96-wellplates. In addition, the assay is ultimately performed at the surface ofthe chip, and the results depend on the kinetics of the hybridization tothe surface, a process that is negatively influenced by steric issuesand diffusion issues. In fact, small microarray chips are notparticularly suited to the detection of rare events, as the diffusion ofthe solution over the chip may not be sufficiently thorough. In order toperform the hybridizations to the microarray chips more efficiently, adedicated fluidics workstation can be used to pump the solution over thesurface of the chip repeatedly; such instruments add cost and time toexecution of the assay.

[0014] The use of spectral barcodes holds great promise for enhancingthe throughput of assays, as described in an application entitled“Semiconductor Nanocrystal Probes for Biological Applications andProcess for Making and Using such Probes,” U.S. Application Ser. No.09/259,982 filed Mar. 1, 1999, the full disclosure of which isincorporated herein by reference. Multiplexed assays may be performedusing a number of probes which include both a spectral label (often inthe form of several semiconductor nanocrystals) and one or moremoieties. The moieties may be capable of selectively bonding to one ormore detectable substances within a sample fluid, while the spectrallabels can be used to identify the probe within the fluid (and hence theassociated moiety). As the individual probes can be quite small, and asthe number of spectral codes which can be independently identified canbe quite large, large numbers of individual assays might be performedwithin a single fluid sample by including a large number of differingprobes. These probes may take the form of quite small beads, with eachbead optionally including a spectral label, a moiety, and a bead body ormatrix, often in the form of a polymer. The reaction times and rareevent identification accuracy of such beads may be quite advantageous,particularly when the beads are free-floating within a fluid, withoutbeing affixed to a surface.

[0015] Together with their substantial advantages, there will besignificant challenges in implementing multiplexed, spectrally encodedbead-based assay techniques. In particular, determining multiplexedassay results by accurately reading each spectral barcode and/or assayresult from within a fluid may prove quite problematic.

[0016] In light of the above, it would generally be desirable to provideimproved systems and methods for sensing and identifying signalgenerating bodies. It would be particularly beneficial if these improvedtechniques facilitated the identification of a plurality of spectralcodes generated by bodies disposed within and/or exposed to a fluid. Totake advantage of the potential capabilities of spectral coding ofmultiplexed assay probes, it would be highly desirable if these enhancedtechniques allowed detection and/or identification of large numbers ofspectral codes and/or other signals in a repeatably, highly timeefficient manner, while providing improved flexibility, ease of use,rare event/condition detection, and/or accuracy.

SUMMARY OF THE INVENTION

[0017] The present invention generally provides improved devices,systems, kits, and methods for detecting and/or identifying a pluralityof spectrally labeled bodies. The invention is particularly well-suitedfor identifying particulate probes or “beads” which have been releasedin a fluid so as to perform a multiplexed assay. These beads can bequite small and may have differing analytical properties, so that theiridentification may be problematic if standard inventory systems areapplied. By spectrally labeling the beads (for example, by includingmaterials which generate identifiable spectra associated with a bead ofa particular type in response to an excitation energy), a plurality ofbeads may be identified within the fluid. Such spectral labels may, forexample, comprise a plurality of differing markers (such assemiconductor nanocrystals, Raman scattering materials, or the like),with the differing markers each generating an identifiable signal, andthe combined signals for each type of probe defining the overallspectrum. These spectrally labeled beads have a wide range ofapplications, and are particularly beneficial for use in multiplexedassay systems.

[0018] The techniques of the present invention will often involvespatially positioning or restraining spectrally labeled bodies such asbeads. The beads may be dynamically restrained by “sweeping” the fluidwith an energy beam such as a focused laser used as an optical tweezers.Alternatively, a plurality of beads may be spatially restrained withinan array of openings. Regardless, it will often be advantageous tooptically image the restrained bead using an optical train having aspectral dispersion element, such as a prism, a refractive grating, atransmissive grating, or the like.

[0019] In a first aspect, the invention provides a spectral labelidentification method comprising spatially restraining a firstspectrally labeled body. A first spectrum is generated from the firstbody while the first body is spatially restrained. The first spectrum isdispersed across a sensor surface, and the first body is identified fromthe first dispersed spectrum.

[0020] The spectrum generating step will often be performed by at leastone semiconductor nanocrystal, often by a plurality of semiconductornanocrystals, and preferably by a plurality of differing semiconductornanocrystal populations. Each semiconductor nanocrystal (or populationthereof) can act as a marker generating a signal having clearly definedsignal characteristics. The combination of these differing markers candefine the label, with the differing signals combining to define theoverall spectrum. When used as assay probes, spectrally encoded bodiesmay also include a moiety such as a selective affinity molecule, amember of a binding pair, or the like. The presence or absence of thespectrally encoded body may comprise an assay result signal, or anadditional assay signal may be generated by interaction of the moietywith an identifiable substance. Typically, a plurality of spectrallylabeled bodies are released in a fluid, and the first body is spatiallyseparated from the other released bodies when the first spectrum isgenerated. Ideally, a plurality of bodies are spatially restrained as anarray in which the bodies are separated from each other to facilitateidentification.

[0021] In many embodiments, a plurality of spectra from the bodies aresimultaneously dispersed across the sensor surface. An array of sitesmay be spaced to avoid excessive overlap of the dispersed spectra sothat each of the bodies can be identified from the associated spectrum.In some embodiments, the spectra may be sensed sequentially with ascanning system by moving a sensor field between the bodies. Suchsequential scanning may be used while simultaneously spatiallypositioning a plurality of bodies, and/or by sequentially restrainingthe bodies.

[0022] The bodies may be restrained within openings in a supportstructure, with the openings sized as to accommodate a single body. Thiscan inhibit confusion by avoiding generating signals with two bodieswhich are in (or near) contact. In some embodiments, the bodies may bedrawn into the openings from the fluids by pumping fluid into theopenings. This allows the bodies to be scanned in a fixed arrayconfiguration, and then expelled to make room for a subsequent array. Inalternative embodiments, the labeled bodies may be sequentiallyspatially restrained by an energy beam, such as by a focused laser beamusing optical tweezing techniques.

[0023] In many embodiments, assay signals may also be sensed from thebodies, the assay signals indicating results of an assay associated withthe bodies. Multiplexed assays using these techniques may make use ofabout 100 different bodies which can be independently identified fromtheir associated spectra, with very highly multiplexed assays oftenincluding at least 1,000 different bodies, optionally being 10,000different bodies.

[0024] In another aspect, the invention provides a method comprisingspatially restraining a plurality of spectrally labeled bodies so as todefine an array. A spectrally dispersed image of the array of bodies isdirected onto a sensor to sense spectra generated by the bodies. Thebodies are identified from the spectra sensed by the sensor.

[0025] The bodies may be restrained within an array of openings affixedin a multi-well plate. The array of bodies may optionally be drawn intothe array of openings by drawing fluid into the openings. The bodies mayalso be expelled from the openings by expelling fluid from the openings.Another array of bodies may then be drawn into the openings by againdrawing fluid into the openings. In other embodiments, the bodies may berestrained in the array by an array of discreet binding sites. Thebinding sites may comprise a material capable of binding to the bodies.Suitable binding materials may comprise a nonspecific “sticky” oradhesive material, a member of a binding pair (with the other memberaffixed to the body), a selective affinity molecule which selectivelybinds to one or more particular bodies, or the like. Regardless ofwhether the array is defined by openings or binding sites, the arraywill preferably be arranged so as to inhibit the presence of more than asingle body at the sites of the array.

[0026] In another aspect, the invention provides a method comprisingreleasing a plurality of bodies in a fluid. A first body is spatiallyrestrained within the fluid by transmitting restraining energy throughthe fluid toward the body. A first spectrum is generated from thespatially restrained first body, and the first body is identified fromthe first spectrum.

[0027] The spatially restraining step may be preformed with a focusedlaser beam, particularly when the laser beam is used as an opticaltweezers. The focused laser beam may be sized and configured to restraina single body. For example, optical tweezers have a “trap” with a sizedetermined at least in part by the geometry of the focus laser beam.Where a size of the spectrally labeled body is at least about half thesize of the trap, the presence of a plurality of beads within the trapwill be inhibited by spatial interference between the beads.

[0028] In some embodiments, the focused laser beam may be configured torestrain a plurality of the bodies simultaneously. For example, the trapmay be elongated so as to restrain the bodies along a line. This may beaccomplished by focusing the laser beam along a line segment.

[0029] In some embodiments, a separate excitation energy may be directedtoward the restrained body, with the body generating the spectrum inresponse to the excitation energy. An alternative embodiments, therestrained body may generate a spectrum in response to the restrainingenergy.

[0030] The spectrum may be transmitted toward a sensor along an opticalpath. The restraining energy may be transmitted toward the body along atleast a portion of the optical path. Optionally, the restrained body maymove within the fluid by moving the restraining energy or the fluid. Therestraining energy may sweep through the fluid to move the body toward afirst site, at which the spectrum may optionally be sensed. Therestraining energy may be swept through the fluid to move a second bodytoward a second site. The transmission of the restraining energy may beinhibited between the first and second sites so as to release the firstbody. Optionally, a series of bodies may be sequentially swept towardthe first site for sequential sensing of their spectra.

[0031] In another aspect, the invention provides a multiplexed assaysystem comprising a support structure having an array of sites. Aplurality of bodies each have a label for generating an identifiablespectrum in response to excitation energy. The bodies are restraininglyreceivable at the sites. An optical train may image at least one site ona sensor surface. The optical train includes a wavelength dispersiveelement.

[0032] In yet another aspect, the invention provides a multiplexed assaysystem comprising a plurality of bodies released in a fluid. The bodieshave labels for generating identifiable spectra. An energy transmitteris coupled to the fluid so as to spatially restrain at least one bodywith a restraining energy beam. A sensor is oriented to reserve thespectrum from the at least one body.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 schematically illustrates an imaging system andhigh-throughput assay method according the principles of the presentinvention.

[0034]FIG. 1A schematically illustrates an exemplary processor for thesystem of claim 1.

[0035]FIG. 2 schematically illustrates probes having spectral labels andassay markers, in which the probes comprise bead structures disposedwithin a test fluid.

[0036] FIGS. 2A-2E schematically illustrate spectral codes or labelshaving a plurality of signals.

[0037]FIG. 3 schematically illustrates a system and method fordetermining a spectrum from a relatively large object by use of anaperture.

[0038]FIG. 4 schematically illustrates a method and structure fordetermining a spectrum from a small object, such as an assay probehaving semiconductor nanocrystal markers, without using an aperture.

[0039]FIGS. 5A and 5B schematically illustrate a system and method fordetermining absolute spectra from a plurality of semiconductornanocrystals by limiting the viewing field with an aperture and byspectrally dispersing the apertured image.

[0040]FIG. 5C schematically illustrates a fluid flow assay scanningsystem and method.

[0041] FIGS. 6A-6C schematically illustrate a plate for positioningsemiconductor nanocrystal assay probes, together with a method for theuse of positioned probes in multiplexed assays.

[0042]FIG. 7 schematically illustrates a method for reading the spectrallabels and/or identifying assay results using the probe positioningplate of FIG. 6C.

[0043]FIG. 8 schematically illustrates an energy beam for spatiallyrestraining and optionally moving a spectrally labeled body, with theenergy being used as an optical tweezers.

[0044]FIG. 9 schematically illustrates a method for using an energy beamto sweep along a surface of a test fluid so as to position one or morespectrally labeled beads.

[0045]FIG. 10 schematically illustrates the use of separate energy beamsfor spatially restraining and generating an identifiable signal of aspectrally labeled body.

[0046]FIG. 11 schematically illustrates dynamically arraying spectrallylabeled beads.

[0047]FIG. 12 schematically illustrates the selective excitation andreading of a spectrally labeled body movably disposed within a testfluid.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0048] The present invention generally provides improved devices,systems, methods, compositions of matter, kits, and the like for sensingand interpreting spectral information. The invention is particularlywell-suited to take advantage of new compositions of matter which cangenerate signals at specific wavelengths in response to excitationenergy. A particularly advantageous signal generation structure for useof the present invention is the semiconductor nanocrystal. Other usefulsignaling structures may also take advantage of the improvementsprovided by the present invention, including conventional fluorescentdies, radioactive and radiated elements and compounds, Raman scatteringmaterials, and the like.

[0049] The invention can allow efficient sensing and/or identificationof a large number of spectral codes, particularly when each codeincludes multiple signals. The invention may also enhance thereliability and accuracy with which such codes are read, and may therebyenable the use of large numbers of spectral codes within a relativelysmall region. Hence, the techniques of the present invention will findadvantageous applications within highly multiplexed assays, inventorycontrol in which a large number of small and/or fluid elements areintermingled, and the like.

[0050] Spectral Labeling

[0051] Referring now to FIG. 1, an inventory system 10 includes alibrary of labeled elements 12 a, 12 b, . . (collectively referred to aselements 12) and an analyzer 14. Analyzer 14 generally includes aprocessor 16 coupled to a detector 18. An energy source 20 transmits anexcitation energy 22 to a sensing field within a first labeled element12 a of library 8. In response to excitation energy 22, first labeledelement 12 a emits radiant energy 24 defining a spectral code. Spectralcode of radiant energy 24 is sensed by detector 18 and the spectral codeis interpreted by processor 16 so as to identify labeled element 12 a.

[0052] Library 8 may optionally comprise a wide variety of elements. Inmany embodiments, labeled elements 12 may be separated. However, in theexemplary embodiment, the various labeled elements 12 a, 12 b, 12 c,. .are intermingled within a test fluid 34. A real imaging is facilitatedby maintaining the labeled elements on or near a surface. As usedtherein, “areal imaging” means imaging of a two-dimensional area. Hence,fluid 34 may be contained in a thin, flat region between planarsurfaces.

[0053] Preferably, detector 18 simultaneously images at least some ofthe signals generated by elements 12 from within a two-dimensionalsensing field. In some embodiments, at least some of the spectralsignals from within the sensing field are sequentially sensed using ascanning system. Regardless, maintaining each label as a spatiallyintegral unit will often facilitate identification of the label. Thisdiscrete spatial integrity of each label is encompassed within the term“spatially resolved labels.” Preferably, the spatial integrity of thebeads and the space between beads will be sufficient to allow at leastsome of the beads to be individually resolved over all other beads,preferably allowing most of the beads to be individually resolved, andin many embodiments, allowing substantially all of the beads to beindividually resolved.

[0054] The spectral coding of the present invention is particularlywell-suited for identification of small or fluid elements which may bedifficult to label using known techniques. Elements 12 may generallycomprise a composition of matter, a biological structure, a fluid, aparticle, an article of manufacture, a consumer product, a component foran assembly, or the like. All of these are encompassed within the term“identifiable substance.”

[0055] The labels included with labeled elements 12 may be adhered to,applied to a surface of, and/or incorporated within the items ofinterest, optionally using techniques analogous to those of standard barcoding technologies. For example, spectral labeling compositions ofmatter (which emit the desired spectra) may be deposited on adhesivelabels and applied to articles of manufacture. Alternatively, anadhesive polymer material incorporating the label might be applied to asurface of a small article, such as a jewel or a component of anelectronic assembly. As the information in the spectral code does notdepend upon the aerial surface of the label, such labels can be quitesmall.

[0056] In other embodiments, the library will comprise fluids (such asbiological samples), powders, cells, and the like. While labeling ofsuch samples using standard bar coding techniques can be quiteproblematic, particularly when a large number of samples are to beaccurately identified, the spectral codes of the present invention canallow robust identification of a particular element from among ten ormore library elements, a hundred or more library elements, a thousand ormore library elements, and even ten thousand or more library elements.

[0057] The labels of the labeled elements 12 will often includecompositions of matter which emit energy with a controllablewavelength/intensity spectrum. To facilitate identification of specificelements from among library 8, the labels of the elements may includecombinations of differing compositions of matter to emit differingportions of the overall spectral code. In other embodiments, the signalsmay be defined by absorption (rather than emission) of energy, by Ramanscattering, or the like. As used herein, the term “markers” encompassescompositions of matter which produce the different signals making up theoverall spectra. A plurality of markers can be combined to form a label,with the signals from the markers together defining the spectra for thelabel.

[0058] The present invention generally utilizes one or more signals fromone or more markers. The markers may comprise semiconductornanocrystals, with the different markers often taking the form ofdifferent particle size distributions of semiconductor nanocrystalshaving different signal generation characteristics. One or more markersmay be combined to form a spectral label which can generate anidentifiable spectrum defining a spectral code, sometimes referred to as“spectral barcodes.” These spectral codes can be used to track thelocation of a particular item of interest or to identify a particularitem of interest.

[0059] In many spectral codes, the different signals will have varyingsignal characteristics which are used as elements of the code. Forexample, semiconductor nanocrystals used in the spectral coding schemecan be tuned to a desired wavelength to produce a characteristicspectral emission or signal by changing the composition and/or size ofthe semiconductor nanocrystal. Additionally, the intensity of the signalat a particular characteristic wavelength can also be varied (optionallyby, at least in part, varying a number of semiconductor nanocrystalsemitting or absorbing at a particular wavelength), thus enabling the useof binary or higher order encoding schemes. The information encoded bythe semiconductor nanocrystals can be spectroscopically decoded from thecharacteristics of their signals, thus providing the location and/oridentity of the particular item or component of interest. As usedherein, wavelength and intensity are encompassed within the term “signalcharacteristics.”

[0060] While spectral codes will often be described herein withreference to the signal characteristics of signals emitted withdiscrete, narrow peaks, it should be understood that semiconductornanocrystals and other marker structures may generate signals havingquite different properties. For example, signals may be generated byscattering, absorption, or the like, and alternative signalcharacteristics such as wavelength range width, slope, shift, or thelike may be used in some spectral coding schemes.

[0061] Semiconductor Nanocrystals

[0062] Semiconductor nanocrystals are particularly well-suited for useas markers in a spectral code system because of their uniquecharacteristics. Semiconductor nanocrystals have radii that are smallerthan the bulk exciton Bohr radius and constitute a class of materialsintermediate between molecular and bulk forms of matter. Quantumconfinement of both the electron and hole in all three dimensions leadsto an increase in the effective band gap of the material with decreasingcrystallite size. Consequently, both the optical absorption and emissionof semiconductor nanocrystals shift to the blue (higher energies) withdecreasing size. Upon exposure to a primary light source, eachsemiconductor nanocrystal distribution is capable of emitting energy innarrow spectral linewidths, as narrow as 20-30 nm, and with a symmetric,nearly Gaussian line shape, thus providing an easy way to identify aparticular semiconductor nanocrystal. The linewidths are dependent onthe size heterogeneity, i.e., monodispersity, of the semiconductornanocrystals in each preparation. Single semiconductor nanocrystalcomplexes have been observed to have full width at half max (FWHM) asnarrow as 12-15 nm. In addition semiconductor nanocrystal distributionswith larger linewidths in the range of 40-60 nm can be readily made andhave the same physical characteristics as semiconductor nanocrystalswith narrower linewidths.

[0063] Exemplary materials for use as semiconductor nanocrystals in thepresent invention include, but are not limited to group II-VI, III-V,and group IV semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe,GaN, GaP., GaAs, GaSb, InP, InAs, InSb, A1S, A1P, A1Sb, PbS, PbSe, Ge,Si, and ternary and quaternary mixtures or alloys thereof. Thesemiconductor nanocrystals are characterized by their nanometer size. By“nanometer” size, it is meant less than about 150 Angstroms (A), andpreferably in the range of 12-150 A.

[0064] The selection of the composition of the semiconductornanocrystal, as well as the size of the semiconductor nanocrystal,affects the signal characteristics of the semiconductor nanocrystal.Thus, a particular composition of a semiconductor nanocrystal as listedabove will be selected based upon the spectral region being monitored.For example, semiconductor nanocrystals that emit energy in the visiblerange include, but are not limited to, CdS, CdSe, CdTe, and ZnTe.Semiconductor nanocrystals that emit energy in the near IR rangeinclude, but are not limited to, InP, InAs, InSb, PbS, and PbSe.Finally, semiconductor nanocrystals that emit energy in the blue tonear-ultraviolet include, but are not limited to, ZnS and GaN. For anyparticular composition selected for the semiconductor nanocrystals to beused in the inventive system, it is possible to tune the emission to adesired wavelength within a particular spectral range by controlling thesize of the particular composition of the semiconductor nanocrystal.

[0065] In addition to the ability to tune the signal characteristics bycontrolling the size of a particular semiconductor nanocrystal, theintensities of that particular emission observed at a specificwavelength are also capable of being varied, thus increasing thepotential information density provided by the semiconductor nanocrystalcoding system. In some embodiments, 2-15 different intensities may beachieved for a particular emission at a desired wavelength, however,more than fifteen different intensities may be achieved, depending uponthe particular application of the inventive identification units. Forthe purposes of the present invention, different intensities may beachieved by varying the concentrations of the particular sizesemiconductor nanocrystal attached to, embedded within or associatedwith an item or component of interest, by varying a Quantum yield of thenanocrystals, by varyingly quenching the signals from the semiconductornanocrystals, or the like. Nonetheless, the spectral coding schemes mayactually benefit from a simple binary structure, in which a givenwavelength is either present our absent, as described below.

[0066] In a particularly preferred embodiment, the surface of thesemiconductor nanocrystal is also modified to enhance the efficiency ofthe emissions, by adding an overcoating layer to the semiconductornanocrystal. The overcoating layer is particularly preferred because atthe surface of the semiconductor nanocrystal, surface defects can resultin traps for electron or holes that degrade the electrical and opticalproperties of the semiconductor nanocrystal. An insulting layer (havinga bandpass layer typically with a bandgap energy greater than the coreand centered thereover) at the surface of the semiconductor nanocrystalprovides an atomically abrupt jump in the chemical potential at theinterface that eliminates energy states that can serve as traps for theelectrons and holes. This results in higher efficiency in theluminescent process.

[0067] Suitable materials for the overcoating layer includesemiconductors having a higher band gap energy than the semiconductornanocrystal. In addition to having a band gap energy greater than thesemiconductor nanocrystals, suitable materials for the overcoating layershould have good conduction and valence band offset with respect to thesemiconductor nanocrystal. Thus, the conduction band is desirably higherand the valence band is desirably lower than those of the semiconductornanocrystal. For semiconductor nanocrystals that emit energy in thevisible (e.g., CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, GaAs) or near IR (e.g.,InP, InAs, InSb, PbS, PbSe), a material that has a band gap energy inthe ultraviolet regions may be used. Exemplary materials include ZnS,GaN, and magnesium chalcogenides, (e.g., MgS, MgSe, and MgTe). Forsemiconductor nanocrystals that emit in the near IR, materials having aband gap energy in the visible, such as CdS, or CdSe, may also be used.While the overcoating will often have a higher bandgap than the emissionenergy, the energies can be, for example, both within the visible range.The overcoating layer may include as many as 8 monolayers of thesemiconductor material. The preparation of a coated semiconductornanocrystal may be found in U.S. patent application Ser. No. 08/969,302filed Nov. 13, 1997, entitled “Highly Luminescent Color-SelectiveMaterials”; Dabbousi et al., J. Phys. Chem B., Vol. 101, 1997, pp. 9463;and Kuno et al., J. Phys. Chem., Vol. 106, 1997, pp. 9869. Fabricationand combination of the differing populations of semiconductornanocrystals may be further understood with reference to U.S. patentapplication Ser. No. 09/397,432, previously incorporated herein byreference.

[0068] It is often advantageous to combine different markers of a labelinto one or more labeled body. Such labeled bodies may help spatiallyresolve different labels from intermingled items of interest, which canbe beneficial during identification. These label bodies may comprise acomposition of matter including a polymeric matrix and a plurality ofsemiconductor nanocrystals, which can be used to encode discrete anddifferent absorption and emission spectra. These spectra can be readusing a light source to cause the label bodies to absorb or emit light.By detecting the light absorbed and/or emitted, a unique spectral codemay be identified for the labels. In some embodiments, the labeledbodies may further include markers beyond the label bodies. Theselabeled bodies will often be referred to as “beads” herein, and beadswhich have assay capabilities may be called “probes.” The structure anduse of such probes, including their assay capabilities, are more fullydescribed in U.S. patent application Ser. No. 09/259,982, previouslyincorporated by reference.

[0069] Fabrication of Labeled Beads

[0070] Referring now to FIG. 2, first and second labeled elements 12 a,12 b within test fluid 34 are formed as separate semiconductornanocrystal probes. Each probe includes a label 36 formed from one ormore populations of substantially mono-disperse semiconductornanocrystals 37. The individual populations of semiconductornanocrystals will often be mono-disperse so as to provide a sufficientsignal intensity at a uniform wavelength for convenient sensing of thevarious signals within the code. The exemplary probes further includeassay markers 38, together with a probe matrix or body material 39,which acts as a binding agent to keep the various markers together in astructural unit. Assay markers 38 generate signals indicating results ofan assay, and are schematically illustrated as semiconductornanocrystals having at least one moiety with selective affinity for anassociated test substance 35 which may be present within sample fluid34. Preparation of the spectrally encoded probes will now be described,followed by a brief description of the use and structure of assaymarkers 38.

[0071] A process for encoding spectra codes into label body materialsusing a feedback system can be based on the absorbance and luminescenceof the semiconductor nanocrystals in a solution that can be used to dyethe materials. More specifically, this solution can be used for encodingof a plurality of semiconductor nanocrystals into a material when thatmaterial is a polymeric bead.

[0072] A variety of different materials can be used to prepare thesecompositions. In particular, polymeric bead materials are an appropriateformat for efficient multiplexing and demultiplexing of finite-sizedmaterials. These label body beads can be prepared from a variety ofdifferent polymers, including but not limited to polystyrene,cross-linked polystyrene, polyacrylic, polysiloxanes, polymeric silica,latexes, dextran polymers, epoxies, and the like. The materials have avariety of different properties with regard to swelling and porosity,which are well understood in the art. Preferably, the beads are in thesize range of approximately 10 nm to 1 mm, more preferably in a sizerange of approximately 100 nm to 0.1 mm, often being in a range from 50nm to 1,000,000 nm, and can be manipulated using normal solutiontechniques when suspended in a solution.

[0073] Discrete emission spectra can be encoded into these materials byvarying the amounts and ratios of different semiconductor nanocrystals,either the size distribution of semiconductor nanocrystals, thecomposition of the semiconductor nanocrystals, or other property of thesemiconductor nanocrystals that yields a distinguishable emissionspectrum, which are embedded into, attached to or otherwise associatedwith the material. The semiconductor nanocrystals of the invention canbe associated with the material by adsorption, absorption, covalentattachment, by co-polymerization or the like. The semiconductornanocrystals have absorption and emission spectra that depend on theirsize and composition. These semiconductor nanocrystals can be preparedas described in Murray et. al., (1993) J. Am. Chem. Soc. 115:8706-8715;Guzelian et. al., (1996) J. Phys. Chem. 100;7212-7219; or InternationalPublication No. WO 99/26299 (inventors Bawendi et al.). Thesemiconductor nanocrystals can be made further luminescent throughovercoating procedures as described in Danek et. al., (1966) Chem. Mat.8(1):173-180; Hines et. al., (1996) J. Phys. Chem. 100:468-471; Peng et.al., (1997) J. Am. Chem. Soc. 119:7019-7029; or Daboussi et. al., (1997)J. Phys. Chem.-B, 101:9463-9475.

[0074] The desired spectral emission properties may be obtained bymixing semiconductor nanocrystals of different sizes and/or compositionsin a fixed amount and ratio to obtain the desired spectrum. The spectralemission of this staining solution can be determined prior to treatmentof the material therewith. Subsequent treatment of the material (throughcovalent attachment, co-polymerization, passive absorption, swelling andcontraction, or the like) with the staining solution results in amaterial having the designed spectral emission property. These spectramay be different under different excitation sources. Accordingly, it ispreferred that the light source used for the encoding procedure be assimilar as possible (preferably of the same wavelength and/or intensity)to the light source that will be used for the decoding. The light sourcemay be related in a quantitative manner, so that the emission spectrumof the final material may be deduced from the spectrum of the stainingsolution.

[0075] A number of semiconductor nanocrystal solutions can be prepared,each having a distinct distribution of sizes and compositions, andconsequently a distinct emission spectrum, to achieve a desired emissionspectrum. These solutions may be mixed in fixed proportions to arrive ata spectrum having the predetermined ratios and intensities of emissionfrom the distinct semiconductor nanocrystals suspended in that solution.Upon exposure of this solution to a light source, the emission spectrumcan be measured by techniques that are well established in the art. Ifthe spectrum is not the desired spectrum, then more of a selectedsemiconductor nanocrystal solution can be added to achieve the desiredspectrum and the solution titrated to have the correct emissionspectrum. These solutions may be colloidal solutions of semiconductornanocrystals dispersed in a solvent, or they may be pre-polymericcolloidal solutions, which can be polymerized to form a matrix withsemiconductor nanocrystals contained within. While ratios of thequantities of constituent solutions and the final spectrum intensitiesneed not be the same, it will often be possible to derive the finalspectra from the quantities (and/or the quantities from the desiredspectra.)

[0076] The solution luminescence will often be adjusted to have thedesired intensities and ratios under the exact excitation source thatwill be used for the decoding. The spectrum may also be prepared to havean intensity and ratio among the various wavelengths that are known toproduce materials having the desired spectrum under a particularexcitation source. A multichannel auto-pipettor connected to a feedbackcircuit can be used to prepare a semiconductor nanocrystal solutionhaving the desired spectral characteristics, as described above. If theseveral channels of the titrator/pipettor are charged or loaded withseveral unique solutions of semiconductor nanocrystals, each having aunique excitation and emission spectrum, then these can be combinedstepwise through addition of the stock solutions. In between additions,the spectrum may be obtained by exposing the solution to a light sourcecapable of causing the semiconductor nanocrystals to emit, preferablythe same light source that will be used to decode the spectra of theencoded materials. The spectrum obtained from such intermediatemeasurements may be judged by a computer based on the desired spectrum.If the solution luminescence is lacking in one particular semiconductornanocrystal emission spectrum, stock solution containing thatsemiconductor nanocrystal may be added in sufficient amount to bring theemission spectrum to the desired level. This procedure can be carriedout for all different semiconductor nanocrystals simultaneously, or itmay be carried out sequentially.

[0077] Once the staining solution has been prepared, it can be used toincorporate a unique luminescence spectrum into the materials of thisinvention. If the method of incorporation of the semiconductornanocrystals into the materials is absorption or adsorption, then thesolvent that is used for the staining solution may be one that issuitable for swelling the materials. Such solvents are commonly from thegroup of solvents including dichloromethane, chloroform,dimethylformamide, tetrahydrofuran and the like. These can be mixed witha more polar solvent, for example methanol or ethanol, to control thedegree and rate of incorporation of the staining solution into thematerial. When the material is added to the staining solution, thematerial will swell, thereby causing the material to incorporate aplurality of semiconductor nanocrystals in the relative proportions thatare present in the staining solution. In some embodiments, thesemiconductor nanocrystals may be incorporated in a different butpredictable proportion. When a more polar solvent is added, afterremoval of the staining solution from the material, material shrinks, orunswells, thereby trapping the semiconductor nanocrystals in thematerial. Alternatively, semiconductor nanocrystals can be trapped byevaporation of the swelling solvent from the material. After rinsingwith a solvent in which the semiconductor nanocrystals are soluble, yetthat does not swell the material, the semiconductor nanocrystals aretrapped in the material, and may not be rinsed out through the use of anon-swelling, non-polar solvent. Such a non-swelling, non-polar solventis typically hexane or toluene. The materials can be separated and thenexposed to a variety of solvents without a change in the emissionspectrum under the light source. When the material used is a polymerbead, the material can be separated from the rinsing solvent bycentrifugation or evaporation or both, and can be redispersed intoaqueous solvents and buffers through the use of detergents in thesuspending buffer, as is well known in the art.

[0078] The above procedure can be carried out in sequential steps aswell. A first staining solution can be used to stain the materials withone population of semiconductor nanocrystals. A second population ofsemiconductor nanocrystals can be prepared in a second stainingsolution, and the material exposed to this second staining solution toassociate the semiconductor nanocrystals of the second population withthe material. These steps can be repeated until the desired spectralproperties are obtained from the material when excited by a lightsource, optionally using feedback from measurements of the interimspectra generated by the partially stained bead material to adjust theprocess.

[0079] The semiconductor nanocrystals can be attached to the material bycovalent attachment, and/or by entrapment in pores of the swelled beads.For instance, semiconductor nanocrystals are prepared by a number oftechniques that result in reactive groups on the surface of thesemiconductor nanocrystal. See, e.g., Bruchez et. al., (1998) Science281:2013-2016; and Ghan et. al., (1998) Science 281:2016-2018, Golvinet. al., (1992) J. Am.

[0080] Chem. Soc. 114:5221-5230; Katari et. al. (1994) J. Phys. Chem.98:4109-4117; Steigerwald et. al. (1987) J. Am. Chem. Soc. 110:3046. Thereactive groups present on the surface of the semiconductor nanocrystalscan be coupled to reactive groups present on the surface of thematerial. For instance, semiconductor nanocrystals which havecarboxylate groups present on their surface can be coupled to beads withamine groups using a carbo-diimide activation step, or a variety ofother methods well known in the art of attaching molecules andbiological substances to bead surfaces. In this case, the relativeamounts of the different semiconductor nanocrystals can be used tocontrol the relative intensities, while the absolute intensities can becontrolled by adjusting the reaction time to control the number ofreacted sites in total. After the bead materials are stained with thesemiconductor nanocrystals, the materials are optionally rinsed to washaway unreacted semiconductor nanocrystals.

[0081] Referring once again to FIG. 2, labeled elements 12 a, 12 b (herein the form of semiconductor nanocrystal probes) may be useful in assaysin a wide variety of forms. Utility of the probes for assays benefitssignificantly from the use of moieties or affinity molecules 35′, asschematically illustrated in FIG. 2, which may optionally be supporteddirectly by label marker 37 of label 36, by the probe body matrix 39, orthe like. Moieties 35′ can have selective affinity for an associateddetectable substance 35, as schematically illustrated by correspondenceof symbol shapes in FIG. 2. The probes may, but need not necessarily,also include an integrated assay marker 38. In some embodiments, theassay marker will instead be coupled to the probes by coupling ofdetectable substance 35 to moiety 35′. In other words, the assay marker38′may (at least initially) be coupled to the detectable substance 35,typically by binding of a dye molecule, incorporation of a radioactiveisotope, or the like. The assay markers may thus be coupled to the probeby the interaction between the moieties 35′ and the test or detectablesubstances 35. In other assays, the assay results may be determined bythe presence or absence of the probe or bead (for example, by washingaway probes having an unattached moiety) so that no dedicated assaymarker need be provided.

[0082] In alternative embodiments, the material used to make the codesdoes not need to be semiconductor nanocrystals. For example, anyfluorescent material or combination of fluorescent materials that can befinely tuned throughout a spectral range and can be excited optically orby other means might be used. For organic dyes, this may be possibleusing a number of different dyes that are each spectrally distinct.

[0083] This bead preparation method can be used generically to identifyidentifiable substances, including cells and other biological matter,objects, and the like. Pre-made mixtures of semiconductor nanocrystals,as described above, are attached to objects to render them subsequentlyidentifiable. Many identical or similar objects can be codedsimultaneously, for example, by attaching the same semiconductornanocrystal mixture to a batch of microspheres using a variety ofchemistries known in the art. Alternatively, codes may be attached toobjects individually, depending on the objects being coded. In thiscase, the codes do not have to be pre-mixed and may be mixed duringapplication of the code, for example using an inkjet printing system todeliver each species of semiconductor nanocrystals to the object. Theuse of semiconductor nanocrystal probes in chemical and/or biologicalassays is more fully described in U.S. patent application Ser. No.09/259,982 filed Mar. 1, 1999, the full disclosure of which isincorporated herein by reference.

[0084] The semiconductor nanocrystal probes of FIG. 2 may also beutilized to detect the occurrence of an event. This event, for example,may cause the source from which energy is transferred to assay marker 38to be located spatially proximal to the semiconductor nanocrystal probe.Hence, the excitation energy from energy source 20 may be transferredeither directly to assay markers 38, 38′, or indirectly via excitationof one or more energy sources adjacent the semiconductor nanocrystalprobes due to bonding of the test substances 35 to the moiety 35′. Forexample, a laser beam may be used to excite a proximal source such as asemiconductor nanocrystal probe 38′ attached to one of the testsubstances 35 (to which the affinity molecule selectively attaches), andthe energy emitted by this semiconductor nanocrystal 38′ may then excitean assay marker 38 affixed to the probe matrix.

[0085] Reading Beads

[0086] Referring once again to FIG. 1, energy source 20 generallydirects excitation energy 22 in such a form as to induce emission of thespectral code from labeled element 12 a. In one embodiment, energysource 20 comprises a source of light, the light preferably having awavelength shorter than that of the spectral code. Energy source 20 maycomprise a source of blue or ultraviolet light, optionally comprising abroad band ultraviolet light source such a deuterium lamp, optionallywith a filter. Energy source 20 may comprise an Xe or Hg UV lamp, or awhite light source such as a xenon lamp or a deuterium lamp, preferablywith a short pass or bandpass UV filter disposed along the excitationenergy path from the lamp to the labeled element 12 so as to limit theexcitation energy to the desired wavelengths. Still further alternativeexcitation energy sources include any of a number of continuous wave(cw) gas lasers, including (but not limited to) any of the argon ionlaser lines (457 nm, 488 nm, 514 nm, etc.), a HeCd laser, a solid-statediode laser (preferably having a blue or ultraviolet output such as aGaN based laser, a GaAs based laser with frequency doubling, a frequencydoubled or tripled output of a YAG or YLF based laser, or the like), anyof the pulsed lasers with an output in the blue or ultraviolet ranges,light emitting diodes, or the like.

[0087] The excitation energy 22 from energy source 20 will inducelabeled element 12 a to emit identifiable energy 24 having the spectralcode, with the spectral code preferably comprising signals havingrelatively narrow peaks so as to define a series of distinguishable peakwavelengths and associated intensities. The peaks will typically have ahalf width of about 100 nm or less, preferably of 70 nm or less, morepreferably 50 nm or less, and ideally 30 nm or less. In manyembodiments, a plurality of separate signals will be included in thespectral code as sensed by sensor 18. As semiconductor nanocrystals areparticularly wellsuited for generating luminescent signals, identifiableenergy 24 from label 12 a will often comprise light energy. To helpinterpret the spectral code from the identifiable energy 24, the lightenergy may pass through one or more monochromator. A Charge-CoupledDevice (CCD) camera or some other two-dimensional detector of sensor 18can sense and/or record the images for later analysis. In otherembodiments, a scanning system may be employed, in which the labeledelement to be identified is scanned with respect to a microscopeobjective, with the luminescence put through a single monochromator or agrating or prism to spectrally resolve the colors. The detector can be adiode array that records the colors that are emitted at a particularspatial position, a two-dimensional CCD, or the like.

[0088] Information regarding these spectra from the labeled elements 12will generally be transmitted as signals sent from sensor 18 toprocessor 16, the processor typically comprising a general purposecomputer. Processor 16 will typically include a central processing unit,ideally having a processing capability at least equivalent to a PentiumI® processor, although simpler systems might use processing capabilitiesequivalent to a Palm® handheld processor or more. Processor 16 willgenerally have input and output capabilities and associated peripheralcomponents, including an output device such as a monitor, an input suchas a keyboard, mouse, and/or the like, and will often have a networkingconnection such as an Ethernet, an Intranet, an Internet, and/or thelike. An exemplary processing block diagram is schematically illustratedin FIG. 1A.

[0089] Processor 16 will often make use of a tangible media 30 having amachine-readable code embodying method steps according to one or moremethods of the present invention. A database 32, similarly embodied in amachine-readable code, will often include a listing of the elementsincluded in library 8, the spectral codes of the labels associated withthe elements, and a correlation between specific library elements andtheir associated codes. Processor 16 uses the information from database32 together with the spectrum characteristics sensed by sensor 18 toidentify a particular library element 12 a. The machine-readable code ofprogram instructions 30 and database 32 may take a wide variety offorms, including floppy disks, optical discs (such as CDs, DVDs,rewritable CDs, and the like), alternative magnetic recording media(such as tapes, hard drives, and the like), volatile and/or nonvolatilememories, software, hardware, firmware, or the like.

[0090] As illustrated in FIG. 1, methods for detecting and classifyingspectral labels (such as encoded materials and beads) may compriseexposing the labels to light of an excitation source so that thesemiconductor nanocrystals of the label are sufficiently excited to emitlight. This excitation source is preferably of an energy capable ofexciting the semiconductor nanocrystals to emit light and may be ofhigher energy (and hence, shorter wavelength) than the shortest emissionwavelength of the semiconductor nanocrystals in the label. Alternativelythe excitation source can emit light of longer wavelength if it iscapable of exciting some of the semiconductor nanocrystals disposed inthe matrix to emit light, such as using two-photon excitation. Thisexcitation source is preferably chosen to excite a sufficient number ofdifferent populations of semiconductor nanocrystals to allow uniqueidentification of the encoded materials. For example, using materialsstained in a 1:2 ratio of red to blue and a 1:3 ratio of red to blue, itmay not be sufficient to only excite the red emitting semiconductornanocrystals, (e.g., by using green or yellow light, of the sample inorder to resolve these beads). It would be desirable to use a lightsource with components that are capable of exciting the blue emittingand the red emitting semiconductor nanocrystals simultaneously, (e.g.,violet or ultraviolet). There may be one or more light sources used toexcite the populations of the different semiconductor nanocrystalssimultaneously or sequentially, but each light source may selectivelyexcite sub-populations of semiconductor nanocrystals that emit at lowerenergy than the light source (to a greater degree than higher energyemitting sub-populations), due to the absorbance spectra of thesemiconductor nanocrystals. Ideally, a single excitation energy sourcewill be sufficient to induce the labels to emit identifiable spectra.

[0091] Spectral Codes

[0092] Referring now to FIGS. 2A-2E, the use of a plurality of differentsignals within a single spectral label can be understood. In this simpleexample, a coding system is shown having two signals. A first signal hasa wavelength peak 40 a at a first discreet wavelength, while a separatesignal has a different wavelength peak 40 b. As shown in FIGS. 2A-2D,varying peak 40 b while the first peak 40 a remains at a fixed locationdefines a first family of spectral codes 1 a through 4 a. Moving thefirst peak 40 a to a new location allows a second family of spectralcodes to be produced, as can be understood with reference to FIG. 2E.

[0093] The simple code system illustrated in FIGS. 2A-2E includes onlytwo signals, but still allows a large number of identifiable spectra.More complex spectral codes having larger numbers of peaks cansignificantly increase the number of codes. Additionally, theintensities of one or more of the peaks may also be varied, therebyproviding still higher order codes having larger numbers of separatelyidentifiable members.

[0094] Spectral Code Reading Systems

[0095] In general, fluorescent labeling is a powerful technique fortracking components in biological systems. For instance, labeling aportion of a cell with a fluorescent marker can allow one to monitor themovement of that component within the cell. Similarly, labeling ananalyte in a bioassay can allow one to determine its presence orabsence, even at vanishingly small concentrations. The use of multiplefluorophores with different emission wavelengths allows differentcomponents to be monitored simultaneously. Applications such as spectralencoding can take full advantage of multicolor fluorophores, potentiallyallowing the simultaneous detection of millions of analytes.

[0096] When imaging samples labeled with multiple chromophores, it isdesirable to resolve spectrally the fluorescence from each discreteregion within the sample. As an example, an assay may be prepared inwhich polymer beads have been labeled with two different chromophoresand the results of the assay may be determined by the ratio of the twotypes of beads within the final sample. One could imagine immobilizingthe beads and counting each of the colors. Electronic imaging mayinvolve a technique for acquiring an image of the sample in whichspectral information is available at each discrete point. While thehuman eye is exceptionally good at distinguishing colors, typicalelectronic photodetectors are often effectively color-blind. As such,additional optical components are often used in order to acquirespectral information.

[0097] Many techniques might be combined with the present invention.Fourier transform spectral imaging (Malik et al. (1996) J. Microsc.182:133; Brenan et al. (1994) Appl. Opt 33:7520) and Hadamard transformspectral imaging (Treado et al. (1989) Anal. Chem 61:732A; Treado et al.(1990) Appl. Spectrosc. 44:1-4; Treado et al. (1990) Appl. Spectrosc.44:1270; Hammaker et al. (1995) J. Mol. Struct. 348:135; Mei et al.(1996) J. Anal. Chem. 354:250; Flateley et al. (1993) Appl. Spectrosc.47:1464); imaging through variable interference (Youvan (1994) Nature369:79; Goldman et al. (1992) Biotechnology 10:1557); acousto-optical(Mortensen et al. (1996) IEEE Trans. Inst. Meas. 45:394; Turner et al(1996) Appl. Spectrosc. 50:277); liquid crystal filters (Morris et al.(1994) Appl. Spectrosc. 48:857); or simply scanning a slit or pointacross the sample surface (Colarusso et al. (1998) Appl. Spectrosc. 52:106A) are methods capable of generating spectral and spatial informationacross a two-dimensional region of a sample.

[0098] Referring now to FIG. 3, a system and method for reading spectralinformation from an arbitrarily large object 50 generally makes use of adetector 52 including a wavelength dispersive element 54 and a sensor56. Imaging optics 58 image object 50 onto a surface of sensor 56.Wavelength dispersive element 54 spectrally disperses the image acrossthe surface of the sensor, distributing the image based on thewavelengths of the image spectra.

[0099] As object 50 is relatively large when imaged upon sensor 56,differentiation of the discreet wavelengths within a spectrum 60 isfacilitated by the use of an aperture 62. As aperture 62 allows only asmall region of the image through wavelength dispersive element 54, thewavelength dispersive element separates the image components based onwavelength alone (rather than on a combination of wavelength andposition along the surface of image 50). Spectra 60 may then be directlydetermined based on the position of the diffracted image upon sensor 56,together with the intensity of image wavelength components as measuredby the sensor.

[0100] Referring now to FIG. 4, a spectra 60 of a spectrally labelednanocrystal bead 64 may be performed using a detector 66 without anaperture. As bead 64 has a signal generating area (as imaged by imagingoptics 58) which is much smaller than a sensing surface of sensor 56,bead 64 can act as a point-source of spectra 60. The various signals ofthe spectral code emanate from the small surface area of the bead, sothat the signal distribution across the sensor surface is dominated bythe wavelength dispersion, and no limiting of the image via an apertureis required. As used herein, a “true point source” is a light sourcewith a dimension which is at least as small as a minimum, diffractionlimited determinable dimension. A light source which is larger than atrue point source may “act” or be “used,” “treated,” or “analyzed” (orthe like) as a point source if it has a dimension or size which issufficiently small that its size acts like an aperture.

[0101] As described above, it will often be advantageous to include aplurality of different spectrally labeled beads within a fluid. Theselabeled beads will often be supported by the surrounding fluid, and/orwill be movable with the fluid, particularly in high-throughputmultiplexed bead-based assays. Optionally, the beads may have a sizesufficient to define a suspension within the surrounding test fluid. Insome embodiments, the beads may comprise a colloid within the testfluid. In some embodiments, beads 64 may be movably supported by asurface of a vessel containing the test fluid, for example, beingdisposed on the bottom surface of the vessel (where probe 64 has adensity greater than that of the test fluid). In other embodiments, thebeads may be affixed to a support structure and/or to each other. Stillfurther alternatives are possible, such as for probe 64 to be floatingon an upper surface of the test fluid, for the bead or beads to beaffixed to or disposed between cooperating surfaces of the vessel tomaintain the positioning of the bead or beads, for the bead or beads tobe disposed at the interface between two fluids, and the like.

[0102] As was described above, it will often be advantageous to includenumerous beads 64 within a single test fluid so as to perform aplurality of assays. Similarly, it will often be advantageous toidentify a large number of fluids or small discreet elements within asingle viewing area without separating out each spectral label from thecombined labeled elements. As illustrated in FIG. 4, the dispersedspectral image 68 of bead 64 upon sensor 56 will depend on both therelative spectra generated by the bead, and on the position of the bead.For example, bead 64′ is imaged onto a different portion 68′ of sensor56, which could lead to misinterpretation of the wavelengths of thespectra if the location of bead 64′ is not known. So long as anindividual bead 64 can be accurately aligned with the imaging optics 58and sensor system 66, absolute spectral information can be obtained.However, as can be understood with reference to FIG. 5A, a plurality ofbeads 64 will often be distributed throughout an area 70.

[0103] To ensure that only beads 64 which are aligned along an opticalaxis 72 are imaged onto sensor 56, aperture 62 restricts a sensing field74 of the sensing system. Where sensor 56 comprises an areal sensor sucha charge couple device (CCD), aperture 62 may comprise a slit apertureso that spectral wavelengths λ can be determined from the position ofthe dispersed images 68 along a dispersion axis of wavelength dispersiveelement 54 for multiple beads 64 distributed along the slit viewingfield 74 along a second axis y, as can be understood with reference toFIG. 5B. Absolute accuracy of the spectral readings will vary inverselywith a width of aperture slit 62, and the number of readings (and hencetotal reading time) for reading all the beads in area 70 will be longeras the slit gets narrower. Nonetheless, the beads 64 within thetwo-dimensional area 70 may eventually be read by the system of FIGS. 5Aand 5B with a scanning system which moves the slit relative to beads 64(using any of a variety of scanning mechanisms, such as movable mirrors,a movable aperture, a flow of the beads passed a fixed aperture, or thelike).

[0104] Sequential sensing of the spectra may be performed by moving theaperture relative to the sensing field, by software, by moving the beads(or other signal sources) relative to the optical train or scanningsystem, or even by scanning one of an excitation energy or the beadsrelative to the other. Aperture scanning may be effected by agalvanometer, by a liquid crystal display (LCD) selective transmissionarrangement, by other digital arrays, or by a digital micro-mirror array(DMD). Bead scanning systems may also use a fluid flow past a slitaperture, with the beads flowing with the fluid. Such bead flow systemsresult in movement of the aperture relative to the beads, even when theaperture remains fixed, as movement may be determined relative to thebead's frame of reference.

[0105] Referring now to FIG. 5C, a simple fluid-flow assay system canmake use of many of the structures and methods described herein above.In the illustrated embodiment, a test fluid 34 flows through a channel131 so that beads 64 move across sensing field 74. Beads 64 within theslit-apertured sensing region are spectrally dispersed and imaged asdescribed above. As the location of the slit-aperture is known, absolutespectral information regarding the label spectra and assay signals maybe determined from dispersed image 68. When a plurality of beads arewithin sensing region 74 but separated along the x axis as shown,multiple beads may be read simultaneously by a CCD, or the like. Flowingof the beads sequentially through sensing region 74 may allowsimultaneous assay preparation and reading using flow injection analysistechniques, or the like.

[0106] Imaging of sensing region 74 may be facilitated by providing athin, flat channel 131 so that beads 64 are near opposed major surfacesof the channel, with at least one of the channel surfaces being definedby a material which is transparent to the spectra and marker signals.This fluid-flow system may be combined with many aspects of the systemsdescribed hereinabove, for example, by providing two different energysources for the label spectra and assay markers, by areal imaging ofbeads 64 distributed throughout a two-dimensional sensing regionadjacent to or overlapping with slit-apertured sensing region 74, andthe like.

[0107] Restrained Position Beads

[0108] Techniques to analyze bead-based assays can be flow based and/orimaging based. In the flow-based analysis, an instrument such as sheathflow cytometer is used to read the fluorescence and scatter informationfrom each bead individually. Flow methods can have the disadvantage ofrequiring a relatively large volume of sample to fill dead volume in thelines and may complicate averaging or re-analysis of data points. Flowmethods allow a large number of beads to be analyzed from a givensample. Imaging based systems, such as the Biometric Image™ system, scana surface to find fluorescence signals. Advantages over the flow systemmay include small (<20 microlitre) sample volumes and the ability toaverage data to improve signal to noise. A potential disadvantage is theuse of a large area in order to keep beads separated, and the dependenceon beads being at an appropriate dilution to ensure that a sufficientnumber can be analyzed without too many forming into doublets, triplets,or the like.

[0109] Referring now to FIGS. 6A-6C, beads can be spatially restrainedand/or immobilized by a support structure 200 such as a planar surface.Typically, beads 64 will be restrained such that they are regularlyspaced in a chosen geometry or array 202. The beads can be immobilizedby restraining the beads in openings 204 in support structure 200, theopenings optionally comprising blind holes or wells. Such wells may befabricated by micromachining wells into the planar surface. For example,7-micron wells that are 7 microns deep, can be created by ablating a 7micron layer of parylene using a focused laser, and by affixing thelayer on a glass surface. Other methods can be used to createmicrostructures on the glass surface that behave as wells, includingelectron beam (or other particle) drilling, mechanical drilling, maskingand plasma etching, and the like.

[0110] The well dimensions may be chosen such that only a single bead iscaptured in the well and such that, when a lateral flow of fluid passesthe beads, the single beads remain trapped in the wells (see FIG. 6C).For example, a 7-micron well may be suitable for analysis of beads fromaround 4 microns to 6 microns, or “monodisperse” 5 micron beads.

[0111] Other methods for capturing and spatially restraining beadsinclude selective deposition of polymers using light-activatedpolymerization, where the pattern of light is determined using aphotoresist. The polymers then bind non-specifically to single beads andother beads can be washed away. More generally, support surface 200 mayoptionally define array 202 as a discreet array of a material capable ofaffixing and/or bonding to beads 64. Suitable array site materials maycomprise a non-specific “sticky” surface, such as those commerciallyavailable from MOLECULAR MACHINES & INDUSTRIES, GMBH, of Heidelberg,Germany. Alternatively, the array material may comprise a specificbinding moiety, a complimentary binding moiety of probes 64 typicallydefining a binding pair with the array material. For example,streptavidin may discreetly deposited on support structure 200 so as todefine array 202, with biotinylated structures disposed on beads 64.Alternatively, streptavidin beads may specifically bind to biotinylatedarray sites of the support structure.

[0112] Regardless of whether array 202 is defined as a series ofopenings 204, or as a series of discreet bead binding sites, it willoften be advantageous to dispose the support structure 200 within afluid container, the support structure typically being affixed within atleast one well of a multi-well plate. In the exemplary embodiment,support structure 200 may comprise a glass structure bonded onto thebottom of at least one well of a multi-well plate. A glass substrate 206of support structure 200 will preferably comprise a relatively thinstructure, typically being thinner than a thickness of a plate materialbordering the well containing the test fluid. By using multi-well plateshaving a clear material bordering the wells, and limiting the thicknessof a substrate 206, reading of beads 64 within openings 204 isfacilitated.

[0113] Layer 208 may be affixed to substrate 206, or may alternativelybe affixed directly to a surface of a multi-well plate, supportstructure 200 typically being deposited along the bottom surface of thewell within a multi-well plate (or other test fluid containing volume)so as to allow gravity to help capture bead 64. Layer 208 may have athickness between about half and one and one half times a size of beads64, with openings 204 also often having a cross-sectional dimensionbetween one half and one and one half times the size of the beads. Layer208 may comprise a material having signal transmission characteristicswhich are significantly different than that of the underlying substrateor container material so as to enhance the accuracy or ease of readingthe beads. Still further structures might be used to immobilize and/orposition the beads, including superparamagnetic bead positioners beingdeveloped by IMMUNICON CORPORATION of Pennsylvania, and by ILLUMINA,INC. of San Diego, Calif.

[0114] In use, the mixture of spectrally encoded beads for a multiplexedassay can be deposited onto the capture surface and allowed to settleinto wells by gravity or to bind to the capture surface. Excess beadsare then washed away leaving single beads filling up some portion, forexample, >90% of the wells or capture positions.

[0115] The captured or spatially restrained beads can then be analyzedusing an imaging system as described above to capture fluorescence dataat various emission wavelengths for each bead. This method providesadvantages over a simple scan of randomly placed beads because (1) beadsare known to be separated so the spatial resolution required fordetection can be reduced (as doublets do not have to be found andrejected). This leads to greater analysis efficiency, (2) the packing ofbeads can be considerably higher while still retaining spatiallyseparated singlet beads, (3) the beads do not move relative to thesupport and so can be scanned multiple times without concerns aboutmovement, and (4) the concentration of beads in the sample does not needto be precise (in the random scattering approach, too high aconcentration can lead to a high packing and eventually a multi-layerstructure, whereas too low a concentration leads to too few beads beinganalyzed).

[0116] In a system where spatial and spectral information are combinedby placing a coarse grating (reflection or transmission) in the emissionpath, such that the emitted light from each bead is spectrally dispersedin one dimension, the use of micromachined wells is particularly useful.The wells can be machined such that the dispersed images of each bead donot overlap. In addition, knowledge of the bead positions means thatabsolute wavelength determination can be carried out rather thanrelative determinations or using a spectral calibrator (See FIG. 7).

[0117] Referring now to FIGS. 6B and 7, array 202 may have a spacing210, 212 between array sites which is selected so as to avoid excessiveoverlap between spectra 214 as distributed across the imaging surface bydispersive element 54. More specifically, a spacing 210 aligned with adispersive axis 216 of dispersive element 54 will preferably besufficient so as to avoid any potential peak-to-peak overlap between thespectra. A spacing 212 transverse to dispersive element 216 may reduced,and/or the array pattern may be staggered so as to increase arraydensity while still avoiding overlap among the spectra. This facilitatescorrelation between the dispersed spectra and the known well/beadposition within array 202, as well as facilitating code interpretation.

[0118] Still further alternative bead positioning means are possible. Inone variation of the positioning wells illustrated in FIGS. 6A-6C, aclosely packed array of collimated holes may be distributed across asurface. Where the holes extend through a substrate defining thesurface, a pressure system may be provided along an opposed surface soas to actively pull beads 64 and test fluid 32 into the array of holes.Such a system would allow a set of beads to be pulled into positioningwells, to have the assay results (optionally including bead labels andassay markers) read from the entrained beads, and then optionally, topush the beads out of the through holes. Such a positioning and readingcycle may be repeated many times to read a large number of beads withina test fluid. While there may be difficulty in transporting the beadsand test fluid to the positioning surface, such a system has significantadvantages.

[0119] Spectrally encoded bodies or beads may be read by a variety ofdiffering systems. Optionally, a confocal excitation source may bescanned across a surface of a sample. Each time the excitation energypasses over an encoded bead, a fluorescence spectrum may be acquired. Byraster-scanning a point excitation source in both the X and Y dimensionsof a surface, all of the beads within a sample may be read sequentially.A disadvantage to such a system is that randomly arrayed beads mightrequire scanning substantially the entire sample with quite high spatialresolution to avoid missing a single bead. This may mean that asignificant amount of time is spent reading the sample surface or volumein regions that do not contain a bead. In addition, once a bead isfound, it should be scanned sufficiently to ensure that the spectrum isread from a similar portion of each bead, for example, from a center ofthe bead. If this is not done with sufficient accuracy, it may bedifficult to determine if a side of a bead, a center of the bead, or thelike has been read, inhibiting accurate assay label quantitation. Suchscanning is time consuming and significantly decreases the efficiency ofa point scanning system. As described above, an array of wells in, forexample, a slide glass may be used to organize the beads. By placing thebeads in an ordered array of wells, it is unnecessary to search for eachbead or to scan the beads carefully to find their centers. Rather, thearray of wells is simply registered to the reader, and the readercollects spectra from the center of each well. This can greatly increasethe rate of scanning, since no time is wasted searching for beads inplaces where they do not exist.

[0120] While the use of arrayed beads is appealing from a speedstandpoint, it may involve removing a sample fluid from a test containerand placing the sample in a specialized reading container structurehaving the wells. It would be highly desirable to be able to read thebeads directly in their original container, such as in the well of a96-well plate to avoid potential sample-to-sample cross contamination.While fabrication or modification of a standard 96-well plate isdescribed above, it may be desirable to provide methods for restrainingand/or removing beads within a fluid. It may also be desirable to have atechnique that did not require one to look for beads in a region of thesample where no beads exist, while still allowing the beads to be readwithin a standard, unmodified 96-well plate or other standard sampleholder.

[0121] As can be understood with reference to FIGS. 8-12, the inventionprovides methods and systems for spatially restraining spectrallylabeled bodies. Optical tweezers can sweep spectrally encoded beads (orany other type of bead) into an ordered array, often as the beads areread. It is not necessary to order the beads prior to reading, as theymay optionally be organized as they are read.

[0122] Referring now to FIG. 8, optical tweezers 220 may comprise alaser beam 222 focused by optics 224 to a tight spot or focus region226. Optical tweezers often include a red or infrared laser beam 222,and may hold a small body, such as a spectrally labeled probe or bead,at or near the center of the point of focus.

[0123] The force used to hold a body using optical tweezers may be lightpressure. Depending on the focal characteristics of optics 224, thesize, intensity, and other characteristics of laser 222, and the like, atrap 228 may be defined by optical tweezers 220, with the trap capableof spatially restraining beads 64 therein. The size of trap 228 may beselected so as to limit the size and/or number of beads 64 which can berestrained therein. For example, any bead smaller than approximately 10μm in diameter that comes in contact with the focused spot 226 may bepulled into the point of highest intensity, as illustrated in FIG. 8. Ifthis point is moved in three-dimensional space, bead 64 may be moved aswell. Such movement of the bead within a moving optical tweezers isencompassed within the term “spatially restrained,” as used herein.However, for beads which are larger than about one half the size of trap228, or about 5 μm in our example, only one bead can exist within trap228 at a time. Optical tweezers are a very standard, and surprisinglysimple tool, used in many different applications. See, e.g. Ashkin(1997) Proc. Natl. Acad. Sci. USA 94: 4853-4860; Helmerson et al. (1997)Clin. Chem: 43:379-383; Quake et al. (1977) Nature (London) 388:151-154;Ashkin (1972) Sci. Amer. 226:63-71; and Ashkin (1970) Phys. Rev. Lett.24:156-159. In the exemplary embodiments, optical tweezers 220 willdefine a trap 228 having a dimension between about 0.1 and two times thesize of bead 64, or between about 100 μm and 100 μm.

[0124] Referring now to FIGS. 9 and 11, optical tweezers 220 may be usedto hold spectrally encoded beads in a moving or fixed position, and/ormay be used to order them in an array 202 for reading. Tweezers 220 maybe focused near a surface 230 along which beads 64 are disposed, such asnear the bottom of a container 232. In the exemplary embodiment, thetweezers are focused near the bottom of a well of a multi-well plate.Beads 64 (optionally, but not necessarily being disposed within fluid34) are moved along surface 232 array sites 236, the beads ideally beingmoved to the center of a detection region of a point-scanning reader.Trap 228 may be scanned along surface 230 by moving optical tweezers220, by moving container 232 relative to the optical tweezers, bymanipulating optics 224 (for example, by moving a scanning mirror with agalvanometer, or the like), or the like.

[0125] Referring now to FIG. 10, excitation energy 22 and spatiallyrestraining energy beam 222 may optionally comprise separate energies,such as a laser beam for spatially restraining beads 64 and a filteredwhite light for exciting semiconductor nanocrystals of beads 64 togenerate an identifiable spectrum. A dichroic mirror 240 may facilitateselectively combing energies having differing wavelengths, with theenergy beams optionally being confocal, so as to define a substantiallysimilar trap and excitation region. In the illustration of FIG. 10, therestraining energy beam and trap 228 are illustrated in solid lines,while the excitation energy and excitation region are schematicallyillustrated with dashed lines.

[0126] Referring now to FIG. 11, moving beads 64 relative to a surface230 so as to provide an ordered array of beads 202 can be understood. Toread all of the beads of an array 202, the scanner may efficiently bedirected toward a first array site 236 and then moved directly to asecond array site, and so forth, skipping the empty space in betweensites. In contrast, if the disordered beads were to be point-scannedprior to ordering the array of FIG. 11, only part of one of the threeillustrated beads 64 would be read, since this bead is not centered onthe detection region or array site 236. This could lead to inaccurateassay results. Furthermore, after reading the first array site, thescanner might miss the remaining beads altogether, since they do notfall directly within the detection regions. Through use of opticaltweezers 220 to order the array of beads, reading of a plurality ofbeads is greatly improved.

[0127] If optical tweezers 220 are energized and oriented at a firstarray site 236, an associated bead partially disposed within the arraysite may be pulled into the center of the trap, thereby providing anaccurate quantitative measure of the assay label bead intensity byaccurately alignment of a scanning system with the bead. After readingthe first bead, the tweezers may be turned off to release the firstbead, and the scanner may advance to the right before the tweezers areagain turned on. If the scanner is moved sufficiently, retrapping of thefirst bead may be inhibited prior to re-energizing the scanner. Once thetweezers are turned on again, the system may be moved to theintermediate spot of the simple array illustrated in FIG. 1 1. While nobead may be initially present in this second array site 236, the processof scanning optical tweezers 220 may pass an adjacent bead and this beadmay be trapped and brought into the intermediate array site. This secondbead can then be read and released as before.

[0128] By using optical tweezers, time which might otherwise be spentlooking between the scanned spots may be used more efficiently. Rather,any beads that fall in between spots may be pulled into a detectionregion or array site. This technique can effectively integrate the areabetween scanned points by bringing any beads that are disposed betweenthe points of an intermittent array scan into the next detection region.Since only one bead may be contained in the trap at a time, such asystem can significantly decrease the reading of multiple beadsco-located at an array site. Conveniently, if multiple beads lie betweenany two points, only one bead may be trapped by the optical tweezers andread. Choosing a scan distance that corresponds to the averageinter-bead spacing can decrease missing beads.

[0129] As can be understood with reference to FIG. 12, an alternativeembodiment may make use of optical tweezers which are separated from thesurfaces of a well. In this embodiment, the detection region remainslocated at the center of the trap. As the tweezers are turned on andoff, the solution is mixed, so that different beads may be brought intothe detection region and held while they are scanned. In other words,movement of fluid 34 relative to the reading system may optionally makeuse of a fixed optical tweezer and bead reading system. This techniquemay allow an identification or tracking system to scan a large number ofbeads without having to resort to precision scanning or control of theconcentration of beads along a bottom or other surface of a well. Hence,such a system may be for simpler and less expensive than a system havingscanning optics or the like. A similar system may restrain beads at areading site within a flow-based system, similar to that of FIG. 5C.

[0130] In other embodiments, the optical tweezers (and morespecifically, the laser beam) may be focused in a single dimension, forexample, to a line (rather than being tightly focused in two dimension,for example, to a point). Such a line-focused laser may create a trapregion that extends along a line, rather than being spherically centeredabout a single point. Such an optical tweezing system may be used tosweep or otherwise spatially restrain beads in distinct lines that canbe scanned by a bead reader system having a slot aperture.

[0131] In many of the optical tweezer systems described above, therestraining energy beam may be an infrared laser, a red laser, or thelike. Optionally, an infrared laser may be used which does not exciteany of the semiconductor nanocrystals within a bead. In otherembodiments, a red laser may be used that simultaneously traps thebeads, and also excites the marker semiconductor nanocrystals so as togenerate the identifiable spectrum. Optionally, the optical readingsystem may make use of at least a portion of the optical train of theoptical tweezers. In other embodiments, the reading and restrainingoptical paths may be separated.

[0132] The above-described spatial restraining optical tweezers, arrayof openings, and the like may be used to form microarrays within avolume or surface of test structure, such as a multi-well plate for avariety of assays. For example, any array-based assay could be processedand detected within a multi-well plate without having to transfer thesample onto an array surface. Detecting signals such as fluorescencefrom such microarrays may be performed using any of the systemsdescribed hereinabove. For example, all of the assays processed for asingle semiconductor nanocrystal pathogen detection might be performedwithin a well of a multi-well plate, with the panel-array printed on thebottom of each well. Related techniques are described in U.S. patentapplication Ser. No. ______, entitled “Single Target Counting AssaysUsing Quantum Dot Nanoparticles” filed Feb. 14, 2001, the fulldisclosure of which is incorporated herein by reference. As part of theprocessing, appropriate analytes may be bound to a surface within amulti-well plate, and the excess analyte removed prior to detection.This may dramatically simplify processing and detection. Other potentialapplications include sandwich immunoassays, DNA/RNA microarrays,surface-based molecular beacon arrays, and the like.

[0133] Specific structures for containing test fluids with beads, and/orfor directing flows of such fluids and beads, may improve spectral codereading performance. Codes may be read from above, from below, or froman angle relative to vertical. Reading codes from below, for example,may be enhanced by using a fluid containing body with an opaque materialover the fluid. The fluid surrounding the beads may have an index ofrefraction which substantially matches that of the material of the lowerportion of the fluid containing body. Such structures may beparticularly beneficial when reading dense bead codes.

[0134] While the exemplary embodiments of the present inventions havebeen described in some detail for clarity of understanding, a variety ofmodifications, adaptations, and changes will be obvious to those ofskill in the art. Hence, the scope of the present invention is limitedsolely by the appended claims.

What is claimed is:
 1. A spectral label identification methodcomprising: spatially restraining a first spectrally labeled body;generating a first spectrum from the first body while the first body isspatially restrained; dispersing the first spectrum from the first bodyacross a sensor surface; and identifying the first body from thedispersed first spectrum.
 2. The method of claim 1, wherein a pluralityof bodies are released in a fluid, and further comprising spatiallyseparating the first body from other released bodies while the firstspectrum is generated.
 3. The method of claim 1, wherein the positioningstep comprises advancing the first body into an opening, the openingsized to accommodate a single body therein.
 4. The method of claim 1,further comprising: spatially restraining a second spectrally labeledbody; generating a second spectrum from the second body whilepositioning the second body, the first spectrum being different than thesecond spectrum; and identifying the second body from the secondspectrum.
 5. The method of claim 4, wherein a plurality of spectrallylabeled bodies are simultaneously spatially restrained at an array ofsites.
 6. The method of claim 5, wherein a plurality of the spectra fromthe bodies are simultaneously dispersed across the sensor surface. 7.The method of claim 6, wherein the array of sites are spaced to avoidexcessive overlap of the dispersed spectra such that each of the bodiescan be identified from the associated spectrum.
 8. The method of claim5, further comprising sequentially sensing the first and second spectrawith a scanning sensor system by moving a sensing field between thebodies.
 9. The method of claim 4, wherein the first and second body aresequentially spatially restrained.
 10. The method of claim 9, furthercomprising drawing the first body into an opening by drawing fluid intothe opening, expelling the body from the first opening, and drawing thesecond body into the opening by drawing fluid into the opening, thesignal generating steps being performed while the first and secondbodies are sequentially disposed within the opening.
 11. The method ofclaim 10, further comprising drawing fluid into an array of openings andexpelling fluid from the array of opening so as to sequentially restraina plurality of arrays of bodies.
 12. The method of claim 9, wherein thefirst and second bodies are spatially restrained by an energy beam. 13.The method of claim 4, further comprising sensing first and second assaysignals from the first and second bodies, each assay signal indicatingresults of an assay associated with the body.
 14. The method of claim 4,further comprising restraining and identifying at least 100 differentbodies from different spectra generated by the bodies.
 15. The method ofclaim 14, further comprising restraining and identifying at least 1000different bodies from different spectra generated by the bodies.
 16. Amethod comprising: spatially restraining a plurality of spectrallylabeled bodies so as to define an array; directing a spectrallydispersed image of the array of bodies onto a sensor to sense spectragenerated by the bodies; identifying the bodies from the spectra sensedby the sensor.
 17. A method as claimed in claim 16, wherein the bodiesare restrained within an array of openings affixed in a multi-wellplate.
 18. A method as claimed in claim 16, further comprising drawingthe array of bodies into the array of opening by drawing fluid into theopenings, expelling the array of bodies from the opening by expellingfluid from the openings, and drawing another array of bodies into thearray of openings by again drawing fluid into the openings.
 19. Themethod of claim 16, wherein the bodies are restrained in the array by anarray of discrete binding sites, the binding sites comprising a materialcapable of binding to the bodies.
 20. A method comprising: releasing aplurality of bodies in a fluid; spatially restraining a first bodywithin the fluid by transmitting restraining energy through the fluidtoward the body; generating a first spectrum from the spatiallyrestrained first body; and identifying the first body from the firstspectrum.
 21. The method of claim 20, wherein the spatially restrainingstep is performed with a focused laser beam, the laser beam acting as anoptical tweezers.
 22. The method of claim 21, wherein the focused laserbeam is sized and configured to restrain a single body.
 23. The methodof claim 22, wherein the focused laser beam defines a trap, and whereina size of the bodies is at least about half the size of the trap so asto inhibit restraining a plurality of the beads within the trap.
 24. Themethod of claim 21, wherein the focussed laser beam is configured torestrain a plurality of the bodies simultaneously.
 25. The method ofclaim 24, wherein the trap is elongated so that the restrained bodiesare arranged along a line.
 26. The method of claim 20, furthercomprising directing excitation energy toward the restrained body, thebody generating the spectrum in response to the excitation energy. 27.The method of claim 20, wherein the restrained body generates thespectrum in response to the restraining energy.
 28. The method of claim20, further comprising transmitting the spectrum toward a sensor alongan optical path, and transmitting the restraining energy toward the bodyalong at least a portion of the optical path.
 29. The method of claim20, further comprising moving the restrained body within the fluid bymoving the restraining energy or the fluid.
 30. The method of claim 29,further comprising sweeping the restraining energy through the fluid tomove the first body toward a first site.
 31. The method of claim 30,further comprising sweeping the restraining energy through the fluid tomove a second body toward a second site.
 32. The method of claim 31,further comprising inhibiting transmission of the restraining energybetween the first and second sites.
 33. The method of claim 30, furthercomprising sweeping the restraining energy through the fluid to move asecond body toward the first site.
 34. A multiplexed assay systemcomprising: a support structure having an array of sites; a plurality ofbodies, each body having a label for generating an identifiable spectrumin response to excitation energy, the bodies being restraininglyreceivable at the sites; and an optical train imaging at least one siteon a sensor surface, the optical train including a wavelength dispersiveelement.
 35. The assay system of claim 34, wherein the sites compriseopenings in the support structure.
 36. The assay system of claim 35,wherein the openings are sized to receive a single body therein so as toseparate the individual bodies for discrete imaging.
 37. The assaysystem of claim 36, wherein the bodies and support structure are exposedto a fluid, and further comprising means for restraining the bodieswithin the openings.
 38. The assay system of claim 37, wherein therestraining means releasably restrains the bodies within the openings,releasing of the bodies allowing the bodies to move with the fluid andout of the openings.
 39. The assay system of claim 35, furthercomprising a pump coupled to the openings for at least one of: drawingfluid and the bodies into the openings, and expelling fluid and thebodies out of the openings.
 40. The assay system of claim 34, whereinthe sites comprise a discrete array of a material capable of bonding tothe bodies.
 41. The assay system of claim 34, wherein the optical traincomprises a scanner for moving a sensing field among the sites.
 42. Theassay system of claim 34, wherein the sites are separated sufficientlyalong a dispersive axis of the dispersive element to avoid excessiveoverlap of dispersed spectra generated simultaneously by the bodies atthe sites.
 43. A multiplexed assay system comprising: a plurality ofbodies released in a fluid, the bodies having labels for generatingidentifiable spectra; an energy transmitter coupled to the fluid so asto spatially restrain at least one body with a restraining energy beam;and a sensor oriented to receive the spectrum from the at least onebody.
 44. The multiplexed assay system of claim 43, wherein the energytransmitter generates a focussed laser beam, energy transmittercomprising an optical tweezers.
 45. The multiplexed assay system ofclaim 43, wherein the at least one body generates the spectrum inresponse to the restraining energy beam.
 46. The multiplexed assaysystem of claim 43, further comprising an excitation energy sourcetransmitting an excitation energy toward the at least one body, the atleast one body generating the spectrum in response to the excitationenergy.
 47. The multiplexed assay system of claim 43, further comprisinga scanner coupled to the restraining energy beam so as to move therestraining energy beam within the fluid.
 48. The multiplexed assaysystem of claim 47, wherein an optical train images the site toward thesensor, the energy transmitter configured to move the at least one bodytoward the site.
 49. The multiplexed assay system of claim 43, furthercomprising an optical train coupling the sensor to the at least onebody.
 50. The multiplexed assay system of claim 49, wherein at least aportion of the optical path also directs the restraining energy beamtoward the at least one body.
 51. The multiplexed assay system of claim43, wherein the restraining energy beam is configured to restrain asingle body.
 52. The multiplexed assay system of claim 5 1, wherein asize of the body is at least about half the size of a trap defined bythe restraining energy beam.
 53. The multiplexed assay system of claim43, wherein the restraining energy beam is configured to restrain aplurality of the bodies along a line.
 54. The multiplexed assay systemof claim 53, wherein an optical train directs a dispersed image of thebodies from along the line onto the sensor surface, the dispersed imagehaving a dispersion axis at an angle to the line.